alexa fluor 680  (Bioss)

Journal: Cellular and Molecular Immunology

Article Title: γδ T-cell autoresponses to ectopic membrane proteins: a new type of pattern recognition

doi: 10.1038/s41423-025-01258-x

Figure Lengend Snippet: Two CDR3δ peptides, GTM and OT3, with tumor binding specificity served as probes to screen the protein ligands of human γδ TCRs. a and b. Immunohistochemical staining of a tumor tissue array with the biotin-labeled GTM peptide a and biotin-labeled OT3 peptide b followed by HRP-conjugated streptavidin. The binding was visualized using DAB as the substrate (brown) (400×). Cancer: tumor tissue; paracancerous: paracarcinoma tissues. Scale bar, 50 μm. GTM and OT3 can specifically bind to several tumor cell lines. Fifty-three types of human tumor cell lines were stained with biotinylated (bio)-GTM c or (bio)-OT3 peptide d and APC-conjugated streptavidin. The binding activity was measured by flow cytometry. The flow data were standardized by the ratio between the mean fluorescence intensity of the peptide conjugate and the secondary antibody alone. The binding strength of the synthetic peptide was compared with that of normal human peripheral blood mononuclear cells (PBMCs). MST analysis of the binding activities of the GTM peptide e and OT3 peptide f to known γδ TCR-recognized stress-inducible ligands. The Kd value for each protein was calculated. g Schematic diagram of OT3- and GTM-γδ CAR molecules. A single-chain Vγ9-Vδ2 or Vγ4-Vδ1 structure, which is composed of a Vδ2 TCR with the tumor-specific CDR3δ sequence OT3 or Vδ1 TCR with the tumor-specific CDR3δ sequence GTM, forms the extracellular region of the γδ CAR. The transmembrane and intracellular segments of a classical third-generation CAR (CD8-CD28-4-1BB-CD3ζ) were linked to the Vγ-Vδ sequence. γδ CARs were cloned and inserted into the lentiviral vector cFUGW. Flow cytometry was used to assess the expression of the activation marker CD69, the secretion of TNF-α h and the cytotoxicity-related molecules granzyme B and perforin i in γδ CAR-T cells after coculture with OVCAR-8 cells in vitro. The statistical data are representative of three independent experiments and are expressed as the means ± SDs. *, p < 0.05; **, p < 0. 01. Cytotoxicity of OT3 CAR-T cells j and GTM CAR-T cells k in different tumor cells in vitro. Cytotoxicity was analyzed with an LDH cytotoxicity detection kit. The data are representative of three independent experiments and are expressed as the means ± SDs. OT3 CAR-αβ T cells exhibit strong cytotoxic activity against tumors in an NSG mouse model. OVCAR-8-transplanted NSG mice were treated with GFP-αβ T cells or OT3 CAR-αβ T cells. Tumor growth in every mouse at each time point was measured by assessing the radiance of the tumor cells l . The tumor growth curve m and survival curve n of each group are shown

Article Snippet: In the OT3 protein microarray, OT3 peptide or biotin, as the primary antibody, was diluted, added, and incubated at 4 °C for 12 h. Cy3-conjugated streptavidin (Bioss) was used to probe the primary antibodies bound to the slide at RT for 1 h. For all the assays, a rabbit anti-GST monoclonal antibody (CST) was added and incubated at RT for 1 h. An Alexa Fluor 647-conjugated goat anti-rabbit IgG (CST) secondary antibody was added and incubated at RT for 1 h. Finally, the microarray slides were scanned with a GenePix 4000B fluorescent microarray scanner (GBI).

Techniques: Binding Assay, Immunohistochemical staining, Staining, Labeling, Activity Assay, Flow Cytometry, Fluorescence, Sequencing, Clone Assay, Plasmid Preparation, Expressing, Activation Assay, Marker, In Vitro

Journal: mBio

Article Title: An inner membrane complex protein IMC1g in Plasmodium berghei is involved in asexual stage schizogony and parasite transmission

doi: 10.1128/mbio.02652-24

Figure Lengend Snippet: The IMCp domain contributed to the IMC targeting of PbIMC1g. ( A ) Schematic drawing of mcherry-tagged full-length and deletion construct of PbIMC1g protein. ( B ) Fluorescent microscopy of mCherry-tagged PbIMC1g (red) and MSP1 (green) in schizonts of the FL, ΔC, and ΔIMCp parasites. The nucleus (nuc) was stained with DAPI (blue). Scale bar, 5 µm. Plot profiles of signal intensities evaluated by Image J software are shown on the right side of each immunofluorescence panel. The mis-localized signals were marked with arrows. ( C ) Solubility assay detecting membrane association of FL (top), ΔC (middle), and ΔIMCp (bottom) using different detergents. Cytosolic soluble proteins are in freeze-thaw (F), integral membrane proteins in Triton X-100 buffer (T), and peripheral membrane proteins in 2% SDS buffer (S). GAPDH, GAPM2, and β-actin were used as loading controls for F-, T-, and S-fractions, respectively. FL-mcherry, 77.9 kDa; ΔC-mCherry, 65.0 kDa; ΔIMCp-mCherry, 59.5 kDa. ( D ) Click chemistry method detecting palmitoylation of PbIMC1g in PbIMC1g HA parasites in the schizont and ookinete stages. Structure and residues for potential palmitoylated cysteine residues of PbIMC1g were shown in the upper PbIMC1g schematic. Localization of PbIMC1g-HA protein on schizont and ookinetes is shown in the lower panel. The alkynyl palmitic acid (Alk-C16) metabolically labeled parasites were stained with Alexa Fluor 488-conjugated streptavidin (Strepv, green), parasite plasma membrane marker MSP1 (cyan) or P25 (cyan), and anti-HA mAb (α-HA, red). Merged images for Strepv, PPM marker, and α-HA are shown in the right column. Scale bar, 5 µm. ( E ) Immunoblot assay of palmitoylated PbIMC1g proteins in ookinetes using CuAAC-click chemistry. The captured palmitoylated proteins labeled with (+) or without (−) Alk-C16 were analyzed by western blotting using anti-HA mAb. S, IP supernatant; P, IP elution. The arrowhead indicates PbIMC1g-HA protein. ( F ) Indirect immunofluorescence microscopy of the PbIMC1g HA parasites at schizont and ookinete stages after treatment with 100 μM 2‐BP. Scale bars, 5 µm. Arrows indicate mis-localized signals of PbIMC1g-HA protein. ( G ) Plot profiles of signal intensities evaluated by Image J software at merozoite and ookinete stages are shown. The mis-localized signals were marked with arrows. ( H ) Pictorial representations of fusion protein constructs used in the experiments are shown. The mutated palmitoylation sites are labeled in the schematics of the Palm-N and Palm-C proteins, respectively. ( I ) Fluorescent microscopy of mCherry-tagged PbIMC1g (red) and MSP1 (green) or P25 (green) at the merozoite and ookinete stages of the Palm-mutN and Palm-mutC parasites. Nuclei (nuc) were stained with DAPI (blue). Scale bar, 5 µm. Plot profiles of signal intensities evaluated by Image J software are shown on the right side of each merozoite stage transgenic strain’s immunofluorescence panel. The mis-localized signals were marked with arrows. ( J ) Solubility assay detects membrane association of Palm-mutN (mutN) and Palm-mutC (mutC) using different detergents. F, freeze-thaw extraction; T, Triton X-100 extraction; S, SDS extraction. GAPDH and GAPM2 were used as loading controls for F- and S-extractions, respectively.

Article Snippet: The cells were then stained with FITC-conjugated streptavidin polyclonal antibody (Thermo Fisher), mouse anti-HA mAb (Abcam), and rabbit anti-MSP1 (1:500) or rabbit anti-P25 (1:500), respectively.

Techniques: Construct, Microscopy, Staining, Software, Immunofluorescence, Solubility, Membrane, Metabolic Labelling, Labeling, Clinical Proteomics, Marker, Western Blot, Transgenic Assay, Extraction

Journal: mBio

Article Title: An inner membrane complex protein IMC1g in Plasmodium berghei is involved in asexual stage schizogony and parasite transmission

doi: 10.1128/mbio.02652-24

Figure Lengend Snippet: Phenotypic analysis of the PvIMC1g TR -transgenic parasites in asexual and sexual stages. ( A ) Schematic figure of predicted palmitoylated sites on PvIMC1g protein. ( B ) Click chemistry method detecting palmitoylation of PvIMC1g-Myc protein in PvIMC1g TR parasites at ookinete stages. The PbIMC1g HA ookinetes were metabolically labeled with (+) or without (−) 25 µM of Alk-C16 for 4 h, biotinylated by click reaction, followed by precipitation with streptavidin agarose resin. The captured palmitoylated proteins were analyzed by western blotting using anti-Myc mAb. S, supernatant; P, pellet. The arrowhead indicates PvIMC1g-Myc protein. ( C ) IFA of 100 µM 2-BP-treated PvIMC1g TR parasites at schizont and ookinete stages. Parasites were co-stained with anti-Myc mAb and anti-MSP1 (schizont) or anti-P25 (ookinetes) sera. The nuclei (nuc) were stained with DAPI. Scale bar, 5 µm. ( D ) Growth curves of the P. berghei ANKA (wild type [WT]) and transgenic PvIMC1g TR (TR) parasites. Each mouse was inoculated with 1 × 10 6 iRBCs, and parasitemia was monitored daily. Data are the mean ± SD of three independent experiments. ( E ) Kaplan-Meier survival curve of mice infected with the WT and TR parasites. Each group has five mice. The graph is representative of three independent experiments. ( F ) Gametocytemia of WT and TR transgenic parasites at 3 dpi. ( G ) Male/female gametocyte ratios of WT and TR parasites at 3 dpi. ( H ) Exflagellation centers formation in the WT and TR parasites at 3 dpi. ( I ) Ookinete conversion rates (%) of the WT and TR parasites. Data were representative from three independent experiments for figures ( F–I ). n = 5 each. ( J ) Prevalence of infection of WT and TR parasite-infected mosquitoes. ( K ) Midgut oocysts in mosquitoes infected with WT and TR strain at 10 dpi. Each dot indicates the oocyst number of a mosquito, while the horizontal bars indicate the mean ± SD. The graph shows two independent feeding experiments. For each feeding experiment, 40 mosquitoes were dissected for the WT and TR parasites, respectively.

Article Snippet: The cells were then stained with FITC-conjugated streptavidin polyclonal antibody (Thermo Fisher), mouse anti-HA mAb (Abcam), and rabbit anti-MSP1 (1:500) or rabbit anti-P25 (1:500), respectively.

Techniques: Transgenic Assay, Metabolic Labelling, Labeling, Western Blot, Staining, Infection